Yersiniae provide a classical experimental model which has been instrumental in resolving mechanisms of microbial virulence. The long-term aims of this proposal are to further exploit this system in order to define the (i) nature of cell-bound microbial processes involved in transport of Fe3+ and the organic iron of hemin, (ii) factors involved in regulating these distinct transport mechanisms, (iii) procedures used to store iron derived from exogenous Fe3+ or hemin, and (iv) relationship of these events to expression of virulence. Specific aims consist of (i) defining surface structures necessary for the siderophore-independent binding of exogenous Fe3+ and hemin, (ii) determining the kinetics of iron transport including binding constants and Km values for uptake of Fe3+ and hemin, (iii) clarifying the roles of variables known to influence the infectivity of yersiniae (temperatures of growth, Mg2+/Ca2+ ratios, and concentration of Fe3+) on regulation of iron uptake and storage, (iv) describing the physical properties and functional characteristics of macromolecules involved in iron storage, (v) resolving the role of the bacteriocin pesticin (an N-acetylglucosaminidase) in converting iron-deprived yersiniae to osmotically stable spheroplasts, and (vi) identifying and comparing the outer membrane receptor for pesticin (and probably hemin) of yersiniae and Escherichia coli. Specialized techniques include preparation of purified outer and inner membranes and their analysis via two-dimensional electropherograms, synthesis of radioactive hemin, and isolation via chromatographic sizing of iron storage components. Accomplishment of these objectives will define the nature of cell-bound siderophore-independent processes of iron transport required by yersiniae (and probably other major pathogenic organisms) for expression of severe systemic disease. Results obtained in these studies will be utilized in designing experiments to determine if injected iron generally enhances virulence by serving as a bacterial nutrient or by interferring with nonspecific mechanisms of host defense (inactivation of antibacterial cationic proteins and scavenging H202 required for 02-dependent killing). Results of these studies will provide a precise understanding of the unique processes of iron transport and storage utilized by major pathogens. No other experimental system presently available can be utilized with equal facility to define these important basic problems.